Introduction
Over the last several decades, dioxins have become the subject of intense scrutiny. This is due to their great toxicity and their assumed widespread presence in the environment. The toxicology of dioxins has been addressed principally through studies of their biological action using animal models and cell culture systems. In addition, the potential threat that dioxins present to human health has been addressed in only a limited manner through epidemiologic studies of populations known to have been exposed to dioxins. The environmental issues have been addressed through the study of the production, release, and degradability of dioxin. Although dioxins have been extensively studied in these ways, their exact mechanism of toxicity in biological systems and their extent of environmental distribution are unknown. This is due in part to the lack of a simplified method of assessing the exposure of biological systems to dioxins and related compounds. The present invention is directed to overcoming this deficiency in the art.
TCDD and Related Chemicals in the Environment
The term dioxin, as commonly used by the news media, is shorthand for 2,3,7,8-tetrachlorodibenzo-p-dioxin ("TCDD"). TCDD is only one member (i.e. congener) of the polychlorinated dibenzo-p-dioxin family, of which there are 75 possible congeners whose structures vary according to the number and location of the chlorine atoms. A source of confusion is that the term "dioxin" is used to indicate either TCDD specifically, or the polychlorinated dibenzodioxin (PCDD) family in general. Biologically, TCDD is the most potent PCDD; most other PCDDs are less active by a factor ranging from ten to thousands. TCDD has been studied most extensively of all the PCDD congeners.
Several aromatic hydrocarbons share biological properties with TCDD, particularly when substituted with chlorine in the lateral positions. The most important of these are the polychlorinated dibenzofurans ("PCDF") and certain members of the polychlorinated biphenyl family ("PCB"). The large number of possible PCDD (75), PCDF (135), and PCB (20) congeners greatly complicates environmental analysis, and complex clean-up procedures are required before such analysis can be undertaken. As used herein, "dioxin-like compounds" includes all members of the above-identified families of compounds and other compounds that induce similar cellular effects, such as azobenzenes and benzopyrenes. TCDD came to scientific and public attention in the early 1970s as a contaminant of the defoliants 2,4,5-trichlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid, notably through forest spraying programs in the U.S. and in Viet Nam.
Prior TCDD Detection Techniques
The principle methods for detection of dioxin-like compounds over the past decade have involved the use of physico-chemical analysis. Such procedures generally involve one or more sample clean-up steps to extract an analyte from the original sample, gas chromatography to separate the analyte of interest from admixture with other compounds, detection by mass spectrometry, and identification of the analyte by comparison to known synthetic standards. These procedures are reviewed in R. E. Clement, "Ultratrace Dioxin and Dibenzofuran Analysis: 30 Years of Advances", Analytical Chemistry, vol. 63, no. 23 (1991) and Analytical News (September/October 1992). Although sensitive, these techniques are not suitable for screening large numbers of samples due to their high cost (i.e., up to $2500 per sample) and the need for the analyses to be carried out at centralized labs by skilled operators. In addition, physico-chemical analysis of dioxin-like compounds is not amenable to the prediction of toxicity of mixtures of dioxin-like compounds or the identification of dioxin-like compounds for which no synthetic standards exist.
Another approach for the detection of dioxin-like compounds has been the use of bioassays in which living cells or animals are dosed with a test sample and then analyzed for the induction of cytochrome P450IA1 activity--a property thought to suggest the presence of dioxin-like compounds.
A number of references disclose cell-line bioassays. In D. E. Tillitt et al., "Characterization of the H4IIE Rat Hepatoma Cell Bioassay as a Tool for Assessing Toxic Potency of Planar Halogenated Hydrocarbons in Environmental Samples," Environ. Sci. Technol., vol. 25, no. 1, pp. 87-92 (1991) utilizes H4IIE rat hepatoma cells to assess the overall toxic potency of various dioxin-like compounds. After such cells were treated with a test sample, they were subjected to spectrofluorometric analysis to detect aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase activity (indicative of cytochrome P450IA1 induction). T. Zacharewski, "Applications of the In Vitro Aryl Hydrocarbon Hydroxylase Induction Assay for determining `2,3,7,8-Tetrachlorodibenzo-p-dioxin Equivalents`; Pyrolyzed Brominated Flame Retardants," Toxicology, 51: 177-89 (1986) ("Zacharewski") is generally similar.
Bioassays requiring live animals have also been utilized. In Zacharewski, rats were injected with test samples and killed. Hepatic microsomal enzyme fractions were recovered and subjected to enzymatic analysis to detect aryl hydrocarbon hydroxylase or ethoxyresorufin-O-deethylase induction.
Although bioassays have been used extensively in the literature, they do not have practicable commercial utility. The chief disadvantage of such assays is their need for live cells or animals, making them unsuitable for a commercial test kit format. In addition, the enzymatic detection of P450IA1 is not very sensitive and requires sophisticated instrumentation.
Due to the above-noted problems associated with physico-chemical analysis and bioassays, increasing attention has been focused on in vitro assays for detection of dioxin-like compounds.
In C. A. Bradfield, et al., "A Competitive Binding Assay for 2,3,7,8-Tetrachlorodibenzo-p-Dioxin and Related Ligands of the Ah Receptor,"Molecular Pharmacology, 34:682-88 (1988), an ammonium sulfate fraction of liver cytosol from C57BL/6J mice was incubated with a test sample and the radioligand [I.sup.125 ]2-iodo-7,8-dibromodibenzo-p-dioxin. Free ligands were then removed by charcoal treatment and bound radioactive ligands were measured with a scintillation counter. This procedure suffers from a number of disadvantages, including the need to obtain a permit for use of radioisotopes and the requirement for equipment to detect and dispose of such radioactive materials. In addition, this assay only measures binding which is not related to toxicity.
In M. Vanderlaan, et al., "Environmental Chemistry--Improvement and Application of an Immunoassay for Screening Environmental Samples for Dioxin Contamination," Environmental Toxicology and Chemistry, vol. 7, pp. 859-70 (1988), levels of dioxin-like compounds in test samples were determined with a direct immunoassay having monoclonal antibodies which bind to the dioxin-like compounds themselves. See also M. Vanderlaan, "ES&T Critical Review--Environmental Monitoring by Immunoassay," Environ. Sci. Technol., vol. 22, no. 3, pp. 247-54 (1988). Such assays are not particularly useful, because they do not distinguish between toxic and non-toxic chemicals all of which may be structurally similar.
U.S. Pat. No. 4,904,595 to Gierthy relates to an epithelial cell line and its use in an in vitro bioassay for dioxin-like activity. Upon exposure to dioxin, a morphological change is induced in the subject XBF cell line, cocultured with lethally irradiated 3T3 cells, to a flat cobblestone appearance, as compared with the fusiform high density state in control cultures not treated with dioxin.
U.S. Pat. No. 4,798,807 to Vanderlaan, et al. discloses monoclonal antibodies which react with dioxin-like compounds and a method of using these antibodies in a sensitive immunoassay for such compounds. These antibodies recognize and bind to dioxin-like compounds in a competitive immunoassay.
U.S. Pat. No. 4,238,472 to Albro, et al. discloses a radioimmunoassay method to detect dioxin-like compounds in environmental samples. This method involves combining a sample containing dioxin (emulsified with detergent) with a first antibody which binds to dioxin and radioactive I.sup.125 labelled dioxin to form an antibody-I.sup.125 -dioxin complex. This causes the labelled and unlabelled dioxin to compete for binding with the antibody. The complex is then reacted with a second antibody to form a precipitate containing dioxin. The radioactivity in the precipitate is assayed, and a curve is utilized to determine the amount of dioxin in the sample
Another approach for detection of dioxin-like compounds is suggested by M. M. Stantostefano, et al., "Effects of Ligand Structure on the In vitro Transformation of the Rat Cytosolic Aryl Hydrocarbon Receptor," Archives of Biochemistry and Biophysics, vol. 297, no. 1, pp. 73-79 (1992). As illustrated in FIG. 4 of that paper, the formation of a dioxin responsive element and a dioxin responsive element binding protein complex is correlated to the concentration of dioxin-like compounds using gel shift analysis. Although this approach is of scientific interest, it is not a suitable commercial format.
In view of the above-noted deficiencies of prior techniques for detecting dioxin-like compounds, the need remains for technology which will accurately detect toxic dioxin-like compounds in a commercially suitable, inexpensive format.